DESCRIPTION: Dr. Franceschi and his colleagues propose to study the molecular mechanisms by which ascorbic acid regulates the differentiation program in osteoblasts. They plan to continue their investigations of the regulation of levels of ascorbic acid in osteoblasts both by studying the AA transport pathway as well as the enzyme responsible for the reduction of dehydroascorbic acid to the active form, ascorbate. They will then determine the effects of specific hormones and drugs on the activities of these two independent determinants of intracellular levels of ascorbate. A second major aim of this proposal focuses on understanding the mechanism by which ascorbate impacts on the transcription of genes encoding bone-specific proteins. Dr. Franceschi and his colleagues have recently shown that the ascorbate effect is likely mediated through secretion of matrix collagen. Their investigation of transcription factors will focus predominantly on basic helix/loop/helix proteins and their interaction with Id proteins. These experiments are predicated on recent observations that a splice variant of Id-1 is down-regulated when osteoblasts are treated with ascorbic acid. Investigators will then use the osteocalcin gene as a model for investigating cis elements that are necessary for induction by ascorbic acid. They plan transfection experiments in which portions of the osteocalcin 5' flank will be attached to a reporter gene in order to define which elements are necessary and sufficient for the ascorbic acid induction. These functional experiments will be supplemented with gel shift and footprinting studies in order to more precisely define functionally important cis elements that bind candidate transcription factors. There will be particular interest in E box sequences that would be putative elements for repression by the Id family of proteins. These studies will enable Dr. Franceschi and his colleagues to gain a better understanding of the mechanisms by which ascorbate affects the programming of bone cell differentiation at the molecular level.